Characterization of a catalase-peroxidase variant (L333V-KatG) identified in an INH-resistant clinical isolate.

catalase-peroxidase (-KatG) is a bifunctional heme-dependent enzyme that has been shown to activate isoniazid (INH), the widely used antibiotic against tuberculosis (TB). The L333V-KatG variant has been associated with INH resistance in clinical isolates from Mexico. To understand better the mechanisms of INH activation, its catalytic properties (catalase, peroxidase, and IN-NAD formation) and crystal structure were compared with those of the wild-type enzyme (WT-KatG).

Engineering a calcium-dependent conformational change in Calbindin D by secondary elements replacement.

EF-hand is a common motif in Ca-binding proteins, some of which present a conformational change upon Ca-binding, a relevant property for signal transduction. In the present work, we investigated the behavior of Calbindin D, a modulator protein with a high affinity for Ca but structurally insensitive to its presence. Its non-canoncal N-terminal EF-hand was replaced by chimeric motifs, containing increasing structural elements from the sensor troponin C SCIII motif.

Engineering Bacillus thuringiensis Cyt1Aa toxin specificity from dipteran to lepidopteran toxicity.

The Cyt and Cry toxins are different pore-forming proteins produced by Bacillus thuringiensis bacteria, and used in insect-pests control. Cry-toxins have a complex mechanism involving interaction with several proteins in the insect gut such as aminopeptidase N (APN), alkaline phosphatase (ALP) and cadherin (CAD). It was shown that the loop regions of domain II of Cry toxins participate in receptor binding.

A reporter system that discriminates EF-hand-sensor motifs from signal-modulators at the single-motif level.

The T-protein is a single-polypeptide bi-functional enzyme composed of a chorismate mutase domain fused to a prephenate dehydrogenase domain (TyrA). We replaced the chorismate mutase domain with canonical or pseudo-Ca(2+)-binding motifs (EF-hand). Canonical-EF-hand-motifs differentiate from pseudo-EF-hand-motifs by experimenting a Ca(2+)-dependent conformational change.

Evolution of a new function in an esterase: simple amino acid substitutions enable the activity present in the larger paralog, BioH.

Gene duplication and divergence are essential processes for the evolution of new activities. Divergence may be gradual, involving simple amino acid residue substitutions, or drastic, such that larger structural elements are inserted, deleted or rearranged. Vast protein sequence comparisons, supported by some experimental evidence, argue that large structural modifications have been necessary for certain catalytic activities to evolve.

The β1 domain of protein G can replace the chorismate mutase domain of the T-protein.

T-protein is composed of chorismate mutase (AroQ(T)) fused to the N-terminus of prephenate dehydrogenase (TyrA). Here, we report the replacement of AroQ(T) with the β1-domain of protein G (Gβ1). The TyrA domain shows a strong dehydrogenase activity within the context of this fusion, and our data indicate that Gβ1-TyrA folds into a dimeric conformation.

Sensitive genome-wide screen for low secondary enzymatic activities: the YjbQ family shows thiamin phosphate synthase activity.

Contemporary enzymes are highly efficient and selective catalysts. However, due to the intrinsically very reactive nature of active sites, gratuitous secondary reactions are practically unavoidable. Consequently, even the smallest cell, with its limited enzymatic repertoire, has the potential to carry out numerous additional, very likely inefficient, secondary reactions.

A modified consensus approach to mutagenesis inverts the cofactor specificity of Bacillus stearothermophilus lactate dehydrogenase.

Lactate dehydrogenase from Bacillus stearothermophilus is specific for NAD+. There have been several attempts to alter the cofactor specificity of this enzyme, but these have yielded enzymes with relatively low activities that still largely prefer NAD+. A modified consensus approach was used to create a library of phylogenetically preferred amino acids situated near the cofactor binding site, and variants were screened for their ability to utilize NMN+.

Increasing the thermal stability of an oligomeric protein, beta-glucuronidase.

The reporter enzyme beta-glucuronidase was mutagenized and evolved for thermostability. After four cycles of screening the best variant was more active than the wild-type enzyme, and retained function at 70 degrees C, whereas the wild-type enzyme lost function at 65 degrees C. Variants derived from sequential mutagenesis were shuffled together, and re-screened for thermostability.